Production of fusion polypeptides has been reported in a number of organisms, including E. coli, yeast, and filamentous fungi. For example, bovine chymosin and porcine pancreatic prophospholipase A.sub.2 have both been produced in A. niger or A. niger var. awamori as fusions to full-length GAI (U.S. Ser. No. 08/318,494; Ward et al., Biotechnology 8:435-440,1990; Roberts et al.,Gene 122:155-161, 1992). Human interleukin 6 (hIL6) has been produced in A. nidulans as a fusion to full-length A. niger GAI (Contreras et al., Biotechnology 9:378-381, 1991). Hen egg white lysozyme (Jeenes et al., FEMS Microbiol. Lett. 107:267-272, 1993) and human lactoferrin (Ward et al., Biotechnology 13:498-503, 1995) have been produced in A. niger as fusions to residues 1-498 of glucoamylase and hIL6 has been produced in A. niger as a fusion to glucoamylase residues 1-514 (Broekhuijsen et al., J. Biotechnol. 31:135-145, 1993). In some of the above experiments (Contreras et al., 1991; Broekhuijsen et al., 1993; Ward et al., 1995) a KEX2 protease recognition site (Lys, Arg) has been inserted between glucoamylase and the desired polypeptide to allow in vivo release of the desired polypeptide from the fusion protein as a result of the action of a native Aspergillus KEX2-like protease.
Additionally, bovine chymosin has been produced in A. niger as a fusion with full-length native alpha-amylase (Korman et al, Curr. Genet. 17:203-212, 1990) and in A. oryzae as a fusion with truncated forms of A. oryzae glucoamylase (either residues 1-603 or 1-511; Tsuchiya et al., Biosci. Biotech. Biochem. 58:895-899, 1994).
A small protein (epidermal growth hormone; 53 amino acids) has been produced in Aspergillus as a tandem fusion of three copies of the protein (U.S. Pat. No. 5,218,093). The trimer of EGF was secreted as a result of the inclusion of an N-terminal secretion signal sequence. However, the EGF molecules were not additionally fused to a protein efficiently secreted by filamentous fungi and no method for subsequent separation of monomeric EGF proteins was provided.
The glaA gene encodes glucoamylase which is highly expressed in many strains of Aspergillus niger and Aspergillus awamori. The promoter and secretion signal sequence of the gene have been used to express heterologous genes in Aspergilli including bovine chymosin in Aspergillus nidulans and A. awamori as previously described (Gwynne, D. et al. (1987) Bio/Technology 5, 713-719 and EPO Publication No. 0 215 594). In the latter experiments, a variety of constructs were made, incorporating prochymosin cDNA, either the glucoamylase or the chymosin secretion signal and, in one case, the first 11 codons of mature glucoamylase. Maximum yields of secreted chymosin obtained from A. awamori were below 15 mg/l in 50 ml shake flask cultures and were obtained using the chymosin signal sequence encoded by pGRG3. These previous studies indicated that integrated plasmid copy number did not correlate with chymosin yields, abundant polyadenylated chymosin mRNA was produced, and intracellular levels of chymosin were high in some transformants regardless of the source of secretion signal. It was inferred that transcription was not a limiting factor in chymosin production but that secretion may have been inefficient. It was also evident that the addition of a small amino terminal segment (11 amino acids) of glucoamylase to the propeptide of prochymosin did not prevent activation to mature chymosin. The amount of extracellular chymosin obtained with the first eleven codons of glucoamylase, however, was substantially less than that obtained when the glucoamylase signal was used alone. Subsequently, it was demonstrated that chymosin production could be greatly increased when a fusion protein consisting of full-length glucoamylase and prochymosin was produced (U.S. Ser. No. 08/318,494; filed Oct. 4, 1994, now U.S. Pat. No. 5,766,934 Ward et al. Bio/technology 8:435-440, 1990).
Aspergillus niger and Aspergillus niger var. awamori (A. awamori) glucoamylases have identical amino acid sequences. The glucoamylase is initially synthesized as preproglucoamylase. The pre and pro regions are removed during the secretion process so that mature glucoamylase is released to the external medium. Two forms of mature glucoamylase are recognized in culture supernatants: GAI is the full-length form (amino acid residues 1-616) and GAII is a natural proteolytic fragment comprising amino acid residues 1-512. GAI is known to fold as two separate domains joined by an extended linker region. The two domains are the 471 residue catalytic domain (amino acids 1-471) and the 108 residue starch binding domain (amino acids 509-616), the linker region being 36 residues in length (amino acids 472-508). GAII lacks the starch binding domain. These details of glucoamylase structure are reviewed by Libby et al. (Protein Engineering 7:1109-1114, 1994) and are shown diagrammatically in FIG. 1.
Trichoderma reesei produces several cellulase enzymes, including cellobiohydrolase I (CBHI), which are folded into two separate domains (catalytic and binding domains) separated by an extended linker region. Foreign polypeptides have been secreted in T. reesei as fusions with the catalytic domain plus linker region of CBHI (Nyyssonen et al., Bio/technology 11:591-595, 1993).